Immunospot s6 entry analyzer

To assay for tumor-specific CTL responses, ELISpot assays were carried out using a Mouse interferon (IFN)-γ single color ELISPOT kit with pre-coated 96-well plates (Cellular Technology Limited) as done previously.^{26 (link)} Splenocytes were adjusted to 5 × 10⁶ cells per mL in CTL test media. ID8-Defb29/Vegf-a-Luc cells and CT26 cells were harvested and resuspended in CTL test media at 5 × 10⁶ cells per mL. 100 μL of the splenocyte suspension was aliquoted into each well (5 × 10⁵ cells per well), and then 100 μL of 5 × 10⁶ cells per L of ID8-Defb29/Vegf-a-Luc cells, 100 μL of 10 μg mL⁻¹ CPMV, 100 μL of 5 × 10⁶ cells per mL of CT26 cells (specificity control), 100 μL of 50 ng mL⁻¹ phorbol 12-myristate 13-acetate (PMA) with 1 mg mL⁻¹ ionomycin (Sigma-Aldrich) (positive control), or 100 mL of CTL test media (negative control) were added. The plates were incubated at 37 °C and 5% CO₂ for 48 h. After incubation, bound cancer cells were gently removed using a stainless-steel spatula and the plates were washed with PBST (0.05% (v/v) Tween-20). Following the manufacturer’s protocol and reagents, the plates were processed to develop IFN-γ spots. The colored spots were quantified using an Immunospot S6 Entry analyzer (Cellular Technology Limited). The splenocytes were evaluated per animal and tested in triplicate for each stimulant.